There is a lack of effective therapeutic strategies to treat Fragile X syndrome (FXS), the most frequent inherited intellectual disability, and the primary monogenic cause of autism. A major challenge for the development of therapies for FXS is to understand the molecular mechanisms leading to the defects in receptor-mediated signaling and protein synthesis that may underlie the broad spectrum of neuronal dysfunctions in FXS. FXS is caused by loss of Fragile X Mental Retardation Protein (FMRP), which binds and translationally regulates many different mRNAs. A critical gap is absence of information on FMRP target mRNAs that are functionally relevant to the global dysregulation of neuronal protein synthesis in FXS. The major objective of the proposed research is to provide evidence that (1) dysregulated expression of the phosphoinositide-3 kinase enhancer (PIKE), a confirmed FMRP target mRNA, is a major mediator of impaired neuronal function in FXS, and that (2) reducing PIKE activity is a promising disease-targeted therapeutic strategy for FXS. Recent studies imply that FMRP is a key regulator of neurotransmitter-mediated PI3K signaling by translationally regulating PI3K signaling components: FMRP was suggested to associate with a few mRNAs coding for components of the PI3K signaling complex, and loss of FMRP leads to increased and dysregulated PI3K activity in mice and patient cells. The PI3K enhancer PIKE is a confirmed FMRP target mRNA within the PI3K complex, and expressed excessively in the absence of FMRP. PIKE is of special interest, because PIKE plays a central role in mediating metabotropic glutamate receptor 1 and 5 (mGlu1/5) - induced activation of PI3K downstream signaling and protein synthesis. Dysregulated mGlu1/5-mediated synaptic plasticity is a hallmark of FXS animal models, and negative modulators of mGlu1/5 receptors are currently in clinical trials with FXS patients; however, these therapies do not considerably ameliorate major phenotypes, such as cognition, probably because they are not targeting the underlying mechanism. The central hypothesis of this proposal is that dysregulated PIKE expression is a key pathomechanism of FXS and underlies phenotypes on the molecular, cellular and behavioral level, including dysregulated mGlu1/5 signaling. This hypothesis will be tested in four specific aims following a comprehensive and multidisciplinary strategy that uses two animal models of FXS, a mouse and a fly model, will employ two different rescue strategies targeting PIKE, genetic Pike knockdown and small peptide inhibitors disrupting mGlu1/5-PIKE-PI3K signaling, and will test phenotypes in four major domains, on the molecular, morphological, electrophysiological and behavioral level. Taken together, the proposed research may not only fill a major gap of knowledge by providing critical information about dysregulated PIKE as a potential mechanism underlying impaired signal transduction in FXS, but may also represent a first step towards developing a novel disease-targeted therapy by testing small peptide inhibitors reducing mGlu1/5-PIKE signaling to ameliorate FXS-associated phenotypes.